1. Field of the Invention
The present invention relates to the field of analytical chemistry and mass spectrometry. This invention specifically relates to sample preparation and purification for analysis by mass spectrometry, particularly matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
2. Background of the Invention
MALDI is widely used for analyzing bio-molecules such as proteins, peptides, nucleic acids and lipids. However, MALDI is sensitive to the presence of interfering materials such as salts, buffers and chaotropes which result in signal suppression. Analysis of microorganisms by mass spectrometry using proteomics approaches has received attention recently due to the speed of analysis. In one approach, proteins from microbes are first extracted, digested using trypsin and purified before loading on to a MALDI target. Therefore, samples to be analyzed are usually cleaned and/or (partially) purified separately, before applying on to the MALDI probe surface. Currently, these cleaning and purification processes typically include chromatographic media and products such as C-18 ZipTips, C-8 ZipTips, and SPE columns. Biomolecules of interest are separated by selectively binding them to hydrophobic or hydrophilic groups attached to the chromatographic media.
Although these off-probe cleaning methods are generally good, there is often some sample loss in the chromatographic media, especially from samples containing low concentrations of analytes. Moreover, these clean up methods are time-consuming and require additional consumables and reagents that can be expensive.
Recently, self assembled monolayer surfaces have been developed as MALDI sample preparation platform and have demonstrated their usefulness in analyzing small volumes of peptides with high levels of contaminants. These techniques incubated the C-18 modified hydrophobic probes in the sample solution for more than 8 hours so as to effectively bind the analytes of interest. On probe cleaning methods by washing with deionized water prior to adding a MALDI matrix to the sample have been shown to significantly increase the protein sequence coverage. However, the long exposure of the probe surface to the sample solution to capture analyte biomolecules is one disadvantage of this method. In another approach, a small volume (1 μL) of the sample for analysis was dried on the probe surface (a procedure which took more than 10 minutes), and then was cleaned by washing the sample on the probe. Although this method appeared to work, the resultant spectra were fairly difficult to obtain since the analyte was localized to a very small region on the probe. As a result only few reasonable single-laser-shot spectra were obtained. This could be potentially explained by realizing that only a small amount of the peptide was bound to the hydrophobic surface due to a small contact area between a near-perfect sphere sample droplet and the hydrophobic surface, since a surface tension angle is close to 180 degrees. As a result the sensitivity of the method suffered due to a small number of analyte molecules binding to the probe surface.
The following articles related to sample preparation and analysis have been reported in the scientific literature, all of which are incorporated herein in entirety by reference:    1. Beavis, R. C. and B. T. Chait, Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc Natl Acad Sci USA, 1990. 87(17): p. 6873-7.    2. Karas, M. and F. Hillenkamp, Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem, 1988. 60(20): p. 2299-301.    3. Ham, B. M., J. T. Jacob, and R. B. Cole, MALDI-TOF MS of phosphorylated lipids in biological fluids using immobilized metal affinity chromatography and a solid ionic crystal matrix. Anal Chem, 2005. 77(14): p. 4439-47.    4. Lim, H., et al., Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry. J Am Soc Mass Spectrom, 2003. 14(9): p. 957-70.    5. Terry, D. E., E. Umstot, and D. M. Desiderio, Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. J Am Soc Mass Spectrom, 2004. 15(6): p. 784-94.    6. Brockman, A. H., B. S. Dodd, and R. Orlando, A desalting approach for MALDI-MS using on-probe hydrophobic self-assembled monolayers. Anal Chem, 1997. 69(22): p. 4716-20.    7. Brockman, A. H., N. N. Shah, and R. Orlando, Optimization of a hydrophobic solid-phase extraction interface for matrix-assisted laser desorption/ionization. J Mass Spectrom, 1998. 33(11): p. 1141-7.    8. Warren, M. E., A. H. Brockman, and R. Orlando, On-probe solid-phase extraction/MALDI-MS using ion-pairing interactions for the cleanup of peptides and proteins. Anal Chem, 1998. 70(18): p. 3757-61.    9. Hooker, J. M., E. W. Kovacs, and M. B. Francis, Interior surface modification of bacteriophage MS2. J Am Chem Soc, 2004. 126(12): p. 3718-9.    10. Strauss, J. H., Jr. and R. L. Sinsheimer, Purification and properties of bacteriophage MS2 and of its ribonucleic acid. J Mol Biol, 1963. 7: p. 43-54.Despite this work, suitable sample preparation techniques are still needed.